Ortho-iodoHoechst 33258 is primarily used in biological research as a fluorescent dye for DNA staining. Its application is significant in cell biology and molecular biology studies, where it helps in visualizing DNA in live and fixed cells. This compound is particularly useful in fluorescence microscopy and flow cytometry to analyze cell cycle stages and DNA content. Additionally, it is employed in studies involving chromatin structure and DNA-protein interactions due to its ability to bind specifically to the minor groove of DNA. Its enhanced fluorescence properties make it a valuable tool for high-resolution imaging and quantitative analysis in various experimental setups.